This invention claims priority of a German patent application DE 100 31 458.9-42 which is incorporated by reference herein.
The invention concerns a scanning microscope having a circulator. Furthermore the invention concerns a confocal scanning microscope with a circulator.
In scanning microscopy, a specimen is illuminated with a light beam in order to observe the reflected light or fluorescent light emitted from the specimen. The focus of the illuminating light beam is generally moved in a specimen plane by tilting two mirrors, the deflection axes usually being perpendicular to one another so that one mirror moves in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, using galvanometer positioning elements; both fast, resonant galvanometers and slower (and more accurate) nonresonant galvanometers are used. The output level of the light coming from the specimen is measured as a function of the position of the scanning beam.
The general construction of a scanning microscope is disclosed in the textbook by James B. Pawley xe2x80x9cHandbook of Biological Confocal Microscopy,xe2x80x9d 1990, Plenum press, New York. The principle of confocal microscopy is described, for example, on pages 4 through 7 (see FIG. 2 in Pawley). In this context, a specimen is scanned using a precisely focused light beam. The light proceeding from the specimen passes through a beam splitter to a detector, in front of which an entrance pinhole is positioned.
In confocal scanning microscopy in particular, a specimen is scanned in three dimensions with the focal point of a light beam. A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto a stop (called the xe2x80x9cexcitation stopxe2x80x9d), a beam splitter, a scanning apparatus for controlling the beam, a microscope optical system, a detection stop, and the detectors for detecting the detected light or fluorescent light. The illuminating light is coupled in via a main beam splitter. The fluorescent light or reflected light coming from the specimen arrives via the same scanning mirror back at the main beam splitter, and passes through the latter and is then focused onto the detection stop, behind which the detectors (usually photomultipliers) are located. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection stop, thus yielding a point datum that results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by recording image data in layers.
Since the wavelengths of the exciting light and detected light differ because of the Stokes shift, dichroic main beam splitters are usually used to observe the fluorescent light. They are designed in such a way that the detected light can pass without hindrance, whereas the illuminating light is deflected at a right angle. No substantial losses occur with this arrangement.
Dichroic beam splitters cannot be used when a specimen (for example one prepared with several dyes) is simultaneously illuminated with light of several wavelengths, and/or when the detected light is polychromatic. Dichroics are not suitable in particular in reflection microscopy, in which the exciting light and detected light have the same wavelength. Broadband beam splitters are used in these situations.
The problems associated with the use of beam splitters in scanning microscopy will now be explained. When the beam splitters used are not polarization beam splitters or chromatic beam splitters, considerable losses of exciting light and/or detected light occur. If a 50:50 beam splitter is used, only 50% of the exciting light arrives at the specimen. Even assuming total reflection at the specimen, of that amount only 50% (i.e. 25% of the illuminating light output) reaches the detector. The detected light loss can be reduced to 10% by using a 90:10 beam splitter, but this entails a loss of 90% of the exciting light. This solution is therefore practical only if sufficient exciting light output is available. There are certain light sources, however, for example blue laser diodes, whose light output is very limited.
It is the object of the invention to eliminate losses of exciting light and/or detected light at the main beam splitter in scanning microscopy. A further object of the invention is for as much as possible of the light generated by the light source to be directed onto the specimen and received by the specimen, in order to limit the light output of the illumination source.
According to the present invention, the object is achieved by a scanning microscope which comprises: at least one illumination source, an objective, at least one detector, and an optical circulator being arranged between the at least one illumination source, the objective, and the at least one detector.
It is a further object of the invention to eliminate losses of exciting light and/or detected light at the main beam splitter in a confocal scanning microscope.
According to the present invention, the object is achieved by a confocal scanning microscope which comprises at least one illumination source, an objective, at least one detector, an optical circulator being arranged between the at least one illumination source, the objective, and the at least one detector, and a detection stop that is arranged in front of the at least one detector.
An advantage of the invention is that the beam splitter is replaced by a circulator. Circulators have been known for some time from microwave technology. Optical circulators are usually magnetooptical components which operate on the basis of the Faraday effect or the Cotton-Mouton effect. One example of a component that is related in principle is the optical isolator, which allows light to pass through in only one direction. Circulators, on the other hand, have not just two but three or more inputs, linked together in circular fashion. In the case of a circulator having inputs 1, 2, and 3, light that is coupled into, for example, input 1 comes back out of input 2. Light that is coupled into input 2 leaves the circulator through input 3.
If, in a microscope arrangement, the illumination light source is then associated with input 1, the specimen with input 2, and the detector with input 3, the circulator then performs the task of the main beam splitter completely and in almost lossless fashion (apart from minor insertion damping).
The reflectivity or transmissivity of beam splitters is generally polarizationdependent. Even high-quality beam splitters cannot be completely optimized, so that differences always occur in reflection and transmission behavior in terms of S and P polarization. If the linear polarization direction of the illuminating light fluctuates, which often happens especially when the light is coupled in with a glass fiber, fluctuations in the illuminating light at the specimen then occur.
Presently available circulators operate independently of polarization, so that these problems do not occur. The data sheets of presently available circulators expressly demonstrate polarization independence.
In particular, fiber-optic circulators can be used. As a particular embodiment, the fiber end at the output associated with the specimen could serve as both the illumination stop and the detection stop. Spectrally broadband circulators are preferably used.